Characterization of fibronectin attachment by a human transitional cell carcinoma line, T24.

The ability of transitional cell carcinoma cells to adhere to and invade the extracellular matrix is important in invasion and metastasis. The glycoprotein fibronectin is associated with laminin and Type IV collagen in the bladder basement membrane. We characterized the interaction between T-24 cells, a cell line derived from an invasive transitional cell tumor, and fibronectin. The cells use the alpha 5 beta 1 heterodimeric integrin receptor complex to mediate adherence via the classical (RGDS) fibronectin binding site. The importance of the alpha 5 beta 1 receptor to T-24 function is unknown and under investigation.

Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis. Amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

Human PMN and monocytes both possess a mechanism for amplifying Fc receptor-mediated phagocytic function, which is dependent on activation of the respiratory burst. The pathway for augmentation of phagocytosis requires superoxide anion, hydrogen peroxide, and lactoferrin and is independent of the hydrogen peroxide-MPO-halide system. In neither cell type is this mechanism induced upon exposure to the opsonized target. PMN require an additional signal for stimulation of the respiratory burst; this is not true of monocytes. On the other hand, monocytes require an exogenous source of lactoferrin in order to activate this pathway for enhanced ingestion. The dependence of this pathway for both PMN and monocytes on superoxide anion, hydrogen peroxide, and cell-bound lactoferrin is consistent with a role for locally generated reactive oxygen metabolites, possibly hydroxyl radicals, in phagocytosis amplification. Patients with chronic granulomatous disease, who are genetically deficient in the ability to activate the respiratory burst, are unable to amplify Fc receptor-mediated phagocytosis. Thus, these patients may have a previously unrecognized defect in the recruitment of phagocytic function at inflammatory sites.

Attachment of mycobacteria to fibronectin-coated surfaces.

This report investigates the extent of the expression of fibronectin (FN) binding properties among the mycobacteria and provides preliminary characteristics of the bacterial molecule(s) mediating attachment. Eight BCG substrains, three Mycobacterium tuberculosis strains and four other mycobacterial species all expressed FN-binding capacity. Treatment of organisms with detergent prior to the binding assay destroyed the FN-binding capacity of BCG but not that of Staphylococcus aureus. Trypsin pretreatment eliminated the FN-binding capacity of both BCG and S. aureus. [35S]Methionine-labelled material in supernatants from BCG and M. tuberculosis cultures attached to FN-coated surfaces. These culture supernatants inhibited the attachment of BCG but not S. aureus to FN-coated surfaces. This inhibitory activity of the supernatants was removed by affinity chromatography on FN-Sepharose but was not affected by similar passage over a control column (human serum albumin attached to Sepharose). These results demonstrate that the ability to bind FN is present in all mycobacterial species tested and suggest that attachment is mediated by trypsin-sensitive cell-surface component(s).

Intravesical Bacillus Calmette-Guérin therapy for murine bladder tumors: initiation of the response by fibronectin-mediated attachment of Bacillus Calmette-Guérin.

Intravesical Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial bladder cancer. Although the mechanisms by which BCG inhibits tumor growth are not known, previous studies have shown that systemic immunization to BCG and the local expression of the immune response in the bladder are associated with a favorable response to BCG therapy. We have investigated the conditions required for the initiation of an immunological response after the intravesical instillation of BCG. Initial histological studies showed that BCG attached to the bladder wall only in areas where the urothelium was damaged by electrocautery and suggested that attachment was associated with the fibrin clot. Quantitative studies verified the histological observations. Minimal BCG attachment (mean less than 10(2) colony forming units) was observed in normal bladders in contrast with a mean of 1.42 X 10(4) colony forming units/bladder in bladders damaged by electrocautery (10 separate experiments). BCG attachment to the bladder wall was durable since organisms were observed in bladders 48 h after instillation. To investigate the proteins to which BCG attached, we tested the binding of BCG to extracellular matrix and inflammatory proteins which comprise a significant portion of the fibrin clot. BCG bound in vitro to coverslips coated in vivo with extracellular matrix proteins but did not bind to control albumin-coated coverslips. BCG also bound to coverslips coated with purified plasma fibronectin but not to coverslips coated with other purified extracellular matrix proteins including laminin, fibrinogen, and type IV collagen. BCG attachment to coverslips coated with either extracellular matrix proteins or purified fibronectin was inhibited by antibodies specific for fibronectin. Moreover, BCG attachment to cauterized bladders in vivo was inhibited by antifibronectin antibodies. These results demonstrate that fibronectin mediates the attachment of BCG to surfaces and suggest that it is the primary component mediating attachment within the bladder. Moreover, the data suggest that the BCG-fibronectin interaction may be a requisite first step for the initiation of the antitumor activity in intravesical BCG for bladder cancer.

Evidence for a conformational change in human fibronectin which occurs between 4 and 37 degrees C.

The effects of temperatures between 4 and 37 degrees C on the structure of human fibronectin (Fn) using monoclonal antibodies (MAb) with specificity for Fn have been studied. Three MAb bound to Fn in solution better at 4 than at 37 degrees C, assessed by both ELISA and immunoprecipitation. These MAb detected temp-dependent conformational changes in Fn which were not associated with a major refolding of the Fn molecule, since the S20,w of Fn and the binding of Fn to polyclonal anti-Fn were unchanged by temp shifts from 4 to 37 degrees C. Fn also was adhered directly to ELISA plates at 4 and at 37 degrees C, and then probed with MAb, with detergents, and with trypsin. One MAb bound better to surface adherent Fn when the Fn had bound to the surface at 4 rather than 37 degrees C. Both SDS and polyoxyethylene (20) sorbitan monolaurate (Tween-20) caused the release of more adherent Fn from surfaces to which it had adhered at 4 degrees C. Finally, trypsin released Fn fragments more rapidly and more completely from surfaces coated at 4 than at 37 degrees C. It is concluded that Fn has different conformations at 4 and 37 degrees C in solution and after adherence to surfaces, as reflected by MAb binding, detergent sensitivity and protease susceptibility. This conformational difference is not associated with alterations in the physiochemical properties of the molecule and is thus a result of a less extensive intramolecular rearrangement than the conformational changes resulting from manipulations of ionic strength or pH, and from binding to collagen or hydrophobic surfaces. Nonetheless, the more subtle conformational change demonstrated here may be important for the biological properties of Fn.

Evidence that the amount of food consumed in early life fixes appetite in the rat.

Rats were raised in litters of 22 (low caloric intake) or litters of 4 (high caloric intake). At the end of 62 wk, rats from large litters were approximately 140 g lighter than those from small litters even though all animals were permitted unrestricted access to food after weaning. One factor responsible for the smaller body size was a lower voluntary food intake after weaning (8,188 +/- 205 g vs. 9,808 +/- 193 g; P less than 0.001). These results provide evidence that the amount of food consumed during suckling plays an important role in determining the habitual food intake of rats in later life. In a separate experiment, rats were raised in litters of 4, 13, 17, or 22. The results show that as litter size increased from 4 to 22, a corresponding reduction in the voluntary intake of food occurred. These results provide evidence that by controlling the food intake of the newborn rat it is possible to "program" the animal for a desired voluntary food intake in later life.

Effect of exercise on hormone-sensitive lipase activity in rat adipocytes.

Hormone-sensitive lipase activity was measured in adipocytes of rats subjected to a 12-wk program of treadmill running. Enzyme activity in the runners sacrificed immediately after exercise increased 2.5-fold (P less than 0.001) in tissue exposed to epinephrine and threefold (P less than 0.001) in tissue not exposed to epinephrine, when the results were expressed per gram of adipose tissue. Increases of almost the same magnitude were observed in runners sacrificed 24 h after their last bout of work. These significant increases in enzyme activity, however, were the result of a significant reduction in the size of cells in the epididymal fat pads of the exercisers compared with those of the freely eating sedentary animals (68.7 +/- 2.7 mum vs. 82.0 +/- 2.7 mum; P less than 0.01). When the results were expressed on a per-cell basis, therefore, hormone-sensitive lipase activity, assayed in the presence or absence of epinephrine, was unaffected by the exercise program. These results provide evidence that the lipolytic capacity of adipocytes of normal, untrained rats is sufficiently large to meet the increased demand for free fatty acids imposed by the exercise program without the need for an adaptive increase in enzyme activity.

Effect of exercise on lipoprotein lipase activity in rat heart and skeletal muscle.

Lipoprotein lipase activity was measured in the three skeletal muscle fiber types of untrained rats and in those of rats subjected to a 12-wk program of treadmill running. Lipoprotein lipase activity in slow-twitch red fibers was approximately 14- to 20-fold higher (P less than 0.001) than that in fast-twitch white and approximately 2-fold higher (P less than 0.001) than that in fast-twitch red fibers in the untrained animals. These results suggest that, in sedentary animals, mainly slow-twitch red and fast-twitch red fibers are capable of taking up plasma triglyceride fatty acids. Regularly performed endurance exercise resulted in significant increase (2- to 4.5-fold) in lipoprotein lipase activity in the three muscle fiber types examined. The increase in lipoprotein lipase activity in response to treadmill running suggests that exercise increases the capacity of these fibers to take up and oxidize plasma triglyceride fatty acids. Cardiac muscle did not undergo an exercise-induced increase in the levels of activity of lipoprotein lipase similar to that seen in skeletal muscle.